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Journal of Scientific and Innovative Research 2016; 5(4): 144-148

Available online at: www.jsirjournal.com

Research Article

ISSN 2320-4818

JSIR 2016; 5(4): 144-148

© 2016, All rights reserved

Received: 21-06-2016

Accepted: 20-07-2016

Tin May Sev

Biotechnology Research

Department, Ministry of Education, Kyaukse, Myanmar

Aye Aye Khai

Professor, Biotechnology Research

Department, Ministry of Education,

Kyaukse, Myanmar

Ayme Aung

Biotechnology Research Department, Ministry of Education,

Kyaukse, Myanmar

San San Yu

Associate Professor, Biotechnology

Research Department, Ministry of Education, Kyaukse, Myanmar

Correspondence: Dr. San San Yu

Associate Professor, Biotechnology

Research Department, Ministry of

Education, Kyaukse, Myanmarr

Evaluation of endophytic bacteria from some rice

varieties for plant growth promoting activities

Tin May Sev, Aye Aye Khai, Ayme Aung, San San Yu*

Abstract

Eighteen endophytic bacteria were isolated from different rice varieties by using Jensen’s nitrogen free

medium. According to morphological and biochemical tests, isolates S1, R1, R2, R5 and R6 may be Rhizobium

spp. and the remaining isolates may be Azospirillum spp. Fourteen out of isolates had plant growth hormone

(Indole Acetic Acid) producing activity. R7 was the best Indole Acetic Acid producer among the isolated

strains at two days incubation period in Jensen’s nitrogen free medium with L-tryptophan 0.5mg /ml at 37˚C

and the concentration produced was 120.55 ppm. In nitrogen free semi-solid agar medium containing trace

amount of bromothymol blue as indicator, all isolates showed nitrogen fixing activity during seven days

incubation period. All isolates except S6 could solubilize phosphate on Pikovskaia’s medium supplemented

with (0.5% Ca3(PO4)2). S5 was the best P- solubilizer at 5 days incubation period both in plate screening

showing zone diameter 45mm and 539.78 ppm quantitatively. Fourteen isolates except (LS, R2, S4, S8) could

also decompose potassium (ranging from 2-30mm in diameter) on K- decomposing medium with 0.12% K-

mica. R3 was the best K-decomposer by plate screening (30mm in diameter) and 19.84 ppm quantitatively. S3

and R3 had good antagonistic activities against Rhizotonia solani and LS and R5 to Phythium spp. and S7 to

Fusarium oxysporum respectively.

Keywords: Endophytic bacteria, Jensen’s nitrogen free medium, Indole Acetic Acid producer, Nitrogen

fixing activity, P-solubilizer, K-decomposer.

INTRODUCTION

Plants are constantly involved in interactions with a wide range of bacteria. These plant associated

bacteria colonize the rhizosphere (rhizobacteria), the phyllosphere (epiphytes) and the inside of plant

tissues (endophytes). Endophytes are sheltered from environmental stresses and microbial competition

by the host plant and they seem to be ubiquitous in plant tissues, having been isolated from flowers,

fruits, leaves, stems, roots and seeds of various plant species [1].

Some endophytic bacteria exert several beneficial effects on host plants, such as stimulation of plant

growth by production of plant hormones [2], nitrogen fixation [3] and induction of resistance to plant

pathogens [4-6], enhanced nutrient uptake, enhanced grain quality and increased drought tolerance. Some

endophytic microorganisms are able to produce compounds of biotechnological value as antibiotics and

antitumor drugs. It is well established that plant bacterial endophytes are to be found in most healthy

plant tissues [6-8]. This particular host endophyte interaction has been variously defined as altruism,

commensalisms, mutualistic, symbiosis or passivity to pathogenicity. Whatever the specific

relationship(s) involved, internal plant colonization by bacteria constitutes a vast and, as yet, little

mapped ecological niche.

There is much debate as to how to define an endophyte. For example, [6] suggested that bacteria and

fungi that are isolated from surface sterilized plant tissues, and that do no apparent harm to the plant,

could be considered as endophytes. Endophytic microorganisms have been defined as those that reside at

some phase of their life cycle within living plant tissues [9, 10]. Endophytes have been found in all plants

that have been examined [11]. These microorganisms include both commensal species, which have no

direct effect on the host plant, and mutualistic symbionts, which could be used in the biological control

of pathogens or plant growth promotion. The intimate relationship between endophytic bacteria and their

host involves co-evolutionary processes, and may influence the physiological mechanisms of plants [12].

145

It has also been shown that in some cases endophytes can accelerate

seed emergence and promote plant establishment under adverse

conditions [13] as well as increase plant growth and hasten plant

development [14]. On the other hand, the effects of endophytes may also

be deleterious, possibly contributing to disease status [15,16], or some

endophytes may interact with other endophytic populations [15]. Causes

of these contrasting outcomes are mostly unclear, but they are very

likely affected by the complex dynamics of interactions among

endophytes, which are in turn affected by environmental conditions,

plant species, and soil type.

MATERIALS AND METHODS

Sampling

The healthy and disease symptomless rice plants were collected from

different rice viz. (Shwe thwe yin, Ma jan taw, Ma naw thukha, Paw san

baykyar and Sin thwe latt) from different places (around Mandalay

Technological University and Kyaukse, Myanmar).

Isolation of Endophytic Bacteria

Endophytic bacteria used in this study were isolated from root and stem

tissues of healthy and disease symptomless rice plants which were

collected from various places. They were washed with tap water to

remove soil and other impurities and then roots and stems were cut into

2-3 cm pieces with sterile surgical blade.

These pieces were dipped and shaked in 96% ethanol for 10 seconds

and then they were rinsed in sterile distilled water for three times.

After that they were dipped and shaked for 10 seconds in 0.1 %

mercuric chloride and rinsed with sterile distilled water for three times.

Then, these sterilized pieces were crushed with sterile motor and pestle.

Finally, the Jensen’s Nitrogen free agar medium was prepared and it

was poured into the sterilized petridish and divided into three parts; one

part for control without inoculation, one for rolling of sterilized tissue

pieces for checking efficient sterilization and one for inoculation of

crushed sample. These were incubated at 37˚C for three to five days.

Screening for Indole Acetic Acid (IAA) Producing Activity

Indole acetic acid (IAA) productions of the isolated strains were

determined by colorimetrically using Salkowski’s reagent (50ml of 35%

perchloric acid: 1ml of 0.5 N FeCl3). To measure IAA qualitatively,

isolated strains were aseptically cultured in Jensen’s nitrogen free broth

containing 0.5 mg L-tryptophan per ml and then incubated at 37ºC for 7

days.

After harvesting, these were centrifuged at 10,000 rpm for 20 mins.

One millilitre of supernatant was mixed with two ml of Salkowski’s

reagent and one drop of orthophosphoric acid and incubated at room

temperature for 25-30 min. Development of pink colour showed IAA

production.

Quantitative Analysis on IAA Production by UV-Vis

Spectrophotometer

Isolated endophytic bacteria were inoculated in Jensen’s nitrogen free

broth containing 0.5 mg L-tryptophan/ml and then incubated at 37°C for

1 to 10 days. After harvesting, these were centrifuged at 10,000 rpm for

20 mins. One ml of supernatant was mixed with 2 ml of Salkowski’s

reagent and one drop of orthophosphoric acid and incubated at room

temperature for 25-30 min.

The absorbance of developed pink colour was measured at 530nm and

then IAA concentrations are calculated by using IAA standard curve

(generated by plotting absorbance on Y-axis and authentic IAA

concentration (ppm) on X-axis).

Screening of Nitrogen-Fixing Activity

All isolates were inoculated in nitrogen free semi-solid agar medium

containing trace amount of BTB as indicator.

Determination of Phosphate Solubilizing Activity by Plate screening

method

A total of 18 bacterial isolates were tested on solid media to select the

most efficient strain. The Pikovskaia's medium was used as a basal

medium and tricalcium phosphate was used as substrate in this study.

After autoclaving, the medium was adjusted to pH 8.6. All isolated

endophytic bacteria were inoculated on this medium and incubated at

37°C for 5 days. After that, the solubilizing activity was measured by

diameter of halo zone (millimetre) around the colony.

Quantitative Determination of Phosphate Solubilizing Activity by

Spectrophotometric Method

The best five phosphorus solubilizing strains out of eighteen isolates

(SR, S5, R6, S7, R8) determined by plate screening method were further

evaluated quantitatively for their P- solubilizing ability in Pikovskaia’s

liquid medium. After autoclaving the medium, it was left to be cool and

0.5 % tricalcium phosphate was added as substrate to avoid its

denaturation by heat. These five strains were inoculated in their

respective broth and incubated at 37˚C and 150 rpm. Uninoculated

medium was used as the control.

Ten-milliliter samples were withdrawn every 24 hours for 1 week and

then each sample was passed through the cation exchange resin. Sodium

molybdate solution and hydrazine sulphate solution were used to form

blue color complex and P- solubilizing activities of these samples were

measured at 830nm by UV-Vis spectrophotometric method. The

intensity of the blue color is propotional to the amount of phosphate

initially corporated in the heteropoly acid.

Determination of Potassium Decomposing Activity by Plate

screening method

The potassium decomposing activity of the isolates was also determined

on K decomposing medium with 0.12% insoluble potassium mica. The

mica sample was washed to remove soluble potassium (K) and dried at

40oC in microwave oven.

All isolated strains were cultured on K decomposing medium

supplemented with insoluble potassium mica and incubated at 37˚C for

5 days. The decomposing activity was measured by diameter of clear

zone around the colony after this incubation period.

Quantitative Determination of K- decomposing Activity

The mica sample was supplemented after cooling the sterilized K-

decomposing broth, and inoculated the best two potassium decomposing

bacteria, S3 and R3, and incubated on shaker at 37˚C and 150 rpm. The

samples were withdrawn every day for one week and the insoluble mica

was removed by centrifugation at 6000 rpm for 10 minutes and then the

supernatants were analyzed by AAS (Atomic Absorption Spectrometry)

method.

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Study on the Antagonistic Activity of Isolated Endophytes

Isolated strains were screened for their antagonistic activities against

common soil borne plant pathogenic fungi like Fusarium oxysporum,

Pythium spp. and Rhizoctonia soloni. This was carried out by hyphal

growth inhibition assay. Czapeak’s medium was used for screening of

antifungal activity. Inhibition of hyphal growth was observed.

Tests for Characterization of Isolated Endophytic Bacteria

These strains were identified on the basis of their colonial morphology,

microscopic characteristics and some biochemical tests etc.

RESULTS AND DISCUSSIONS

Isolation and Identification of Rice Endophytic Bacteria

Eighteen endophytic bacteria were isolated from different rice viz.

(Shwe thwe yin, Ma jan taw, Ma naw thukha, Paw san bay kyar and Sin

thwe latt) by using Jensen’s nitrogen free medium.

Colonial morphologies of all isolates were round raised, mucoid and

transparent on Jensen’s nitrogen free medium. All isolates were gram-

negative and cell diameters are in the range of 0.5-1.5 x 2-4 µm rods.

According to morphological and biochemical tests, isolates S1, R1, R2,

R5 and R6 may be Rhizobium spp. and the remaining isolates may be

Azospirillum spp.

Although Rhizobium association had been extensively explored in the

root nodules of legumes where they fix atmospheric nitrogen, recent

studies also suggest that Rhizobium can exhibit plant growth promoting

(PGP) activities with non-legumes such as rice [17].

Azospirillum spp have broad host range and these two species can

promote plant growth via secretion of plant growth hormones (IAA,

GA), vitamins, solubilization of insoluble phosphates, decomposition of

potassium, biological nitrogen fixation, induced resistance to some plant

pathogens via secretion of antimicrobial compounds that may hinder

colonization of hosts by phytopathogens, thereby suppressing the

diseases they cause, enhancement in stress resistance to abiotic factors [18] such as salt tolerance, drought resistance and production of

siderophores under iron limiting condition.

Figure 1: Growth of Endophytic Bacteria on Jensen’s medium (Azospirillum

spp.) and on YEM Medium (Rhizobium spp.)

Figure 2: Microscopic Morphology of Some Isolated Endophytes

Table 1: Biochemical Characteristics of Isolated Endophytic Bacteria

R3, R7, R8, S2, S3, S4, S5,

S6, S7, S8, LS, SS, SR

S1, R1, R2, R5, R6

Gram reaction - -

Colonial morphology

Small, white, dense

colonies on semi-

solid NFb agar

medium

Circular, convex,

mucous, opaque

or transparent on

YEM

Microscopic

morphology

Clump shape, slightly

curved and straight

rod, single and pairs

(0.6–1.7 x 2.1–

3.8µm)

Small rod, x, y

shape

(0.5–1 x 1.2–3

µm)

Motility + +

Gelatin agar - -

Starch hydrolysis - -

Catalase + +

Citrate utilization + -

Carbon sources utilization

Mannitol + +

Glucose + +

Sucrose + +

Screening on Indole Acetic Acid (IAA) Producing Activity

Four isolates, R2, SS, SR, LS couldn’t produce IAA by screening them

with IAA detecting reagent (Salkowski’s reagent) after 7 days

incubation.

Thus, fourteen out of isolates had plant growth hormone, IAA,

producing activity determined by the development of pink colour.

Figure 3: Screening on IAA Producing Activities of Fourteen Isolates

Quantitative Determination of IAA Producing Activity of Isolated

Endophytic Bacteria

Among fourteen isolates, R7 was the best IAA producer and the

concentration produced was 120.55 ppm in two days incubation period.

S6 produced 104.58 ppm and R8 produced 95.55 ppm. R6 had the lowest

C R1 S4 R5 S6 R7 S8 S5 R8 S3 R3 S7 S2 S1 R6C R1 S4 R5 S6 R7 S8 S5 R8 S3 R3 S7 S2 S1 R6

Biochemical

characteristics

Isolated

Bacteria

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IAA producing activity among fourteen isolates, and it produced 6.35

ppm in 2 days incubation period.

The highest concentration of IAA was detected in two days incubation

period and in the following days, these IAA hormones may change to

other compounds and so these cannot be detected by the IAA detecting

reagent (Salkowski’s reagent).

Figure 4: The Best Incubation Periods for High IAA Productivity of Isolated

Endophytic Bacteria (0.5mg L- tryptophan/ml)

Nitrogen Fixation Activity of Isolated Endophytic Bacteria

In nitrogen free semi-solid agar medium containing trace amount of

bromothymol blue as indicator, all isolates showed nitrogen fixing

activity after three days incubation period.

Figure 5: Nitrogen-Fixing Activity of Thirteen Azospirillum spp. In Semi-solid

Nitrogen Free Broth during Seven Days Incubation Period

Figure 6: Nitrogen-Fixing Activity of Five Rhizobium spp. in Semi-solid

Nitrogen Free Broth during Seven Days Incubation Period

Results for Phosphate Solubilizing Activity of Isolated Endophytic

Bacteria

All isolates except S6 could solubilize phosphate ranging from 3-45 mm

in diameter on Pikovskaia’s medium supplemented with (0.5%

Ca3(PO4)2) and S5 is the best P- solubilizer. In plate screening, halo

zone of S5 was 45 mm in diameter and also the best P solubilizer by

spectrophotometric method, and solubilized P concentration was 539.78

ppm at 5 days incubation period. SR was second best P solubilizing

strain and solubilized P concentration was 485.55 ppm and S7 had the

lowest P solubilizing activity among five strains.

Table 2: Soluble P Concentration of Five Isolates (ppm) in Pikovskaia’s Medium

by Spectrophotometric Method (at 830nm)

Isolates Incubation Periods (Days)

S5 269.54 539.78 152.74

S7 76.16 127.49 98.42

R6 62.5 157.3 102.05

R8 84.84 236.77 81.96

SR 121.28 485.55 252.57

Potassium Decomposing Activity of Isolated Endophytic Bacteria

Fourteen isolates except (LS, R2, S4, S8) could decompose potassium

(ranging from 2-30 mm in diameter) on K- decomposing medium with

0.12% K- mica and R3 was the best K-decomposer by plate screening

method and by AAS method (19.84 ppm).

Table 3: Soluble K Concentration (ppm) Decomposed by Two

Endophytic Strains in 3, 5 and 7 Days Incubation Periods by AAS

Method

Isolates Incubation Period (Days)

3 5 7

S3 3.36 14.91 18.73

R3 3.28 17.46 19.84

Results for Antagonistic Activity

S3 and R3 had good antagonistic activities against Rhizotonia solani and

LS and R5 against Phythium spp. and S7 to Fusarium oxysporum

respectively. They couldn’t be overwhelmed by these plant pathogens

and could live with these pathogens. So, this fact can benefit the host

plants they lived.

CONCLUSION

In this study, eighteen endophytic bacteria were isolated from stems and

roots of different rice varieties from different places. According to the

biochemical tests, five isolates were assumed to be Rhizobium spp. and

thirteen isolates were Azospirillum spp. although species identification

could not be done.

Most strains show high IAA producing activity at 2 days incubation

period by UV-VIS spectrophotometer method (530nm) and isolate R7

was found to be the best strain producing IAA, 120.55 ppm. In

analyzing nitrogen fixation activity by ammonium test kit, S5, S7, S8

isolates could fix nitrogen little and others couldn’t fix. In nitrogen free

semi-solid agar medium containing trace amount of bromothymol blue

as indicator, all isolates showed nitrogen-fixing activity during seven

days incubation period.

According to the results of phosphorous solubilizing activity by plate

screening method and by UV-Vis spectrophotometric method (830 nm),

isolate S5 was found to be the best phosphate solubilizer. R3 is the best

K-decomposer by plate screening method and by AAS method. S3 and

R3 have good antagonistic activities against Rhizotonia solani and LS

and R5 against Phythium spp. and S7 to Fusarium oxysporum

respectively. They couldn’t be overwhelmed by these plant pathogens

and could live with these pathogens. So, this fact can benefit the host

plants.

148

Acknowledgements

I would like to thank my supervisor, Dr. Aye Aye Khai and Dr. San San

Yu, Biotechnology Research Department, Kyaukse, for their generous

supports and helpful discussions throughout my research work. I also

deeply thank to my colleagues at our department for their kindly help

and suggestions.

REFERENCES

1. Kobayashi, D. Y. and Palumbo, J. D.: Bacterial endophytes and their

effects on plants and uses in agriculture, In: Bacon, C.W. and White, J. F.

(Eds.) Microbial endophytes. Marcel Dekker, Inc., N.Y., New York, 2000,

pp. 199-233.

2. Sturz, A. V., Christie, B. R., Matheson, B. G. and Nowak, J.: Biodiversity

of endophytic bacteria which colonize red clover nodules, roots, stems and

foliage and their influence on host growth, Biol. Fertil. Soils, 1997;25:13 –

19.

3. Kirchhorf, G., Reis, V. M., Baldani, J. I., Eckert, B., Döbereiner, J. and

Hartmann, A.: Occurrence, physiological and molecular analysis of

endophytic diazotrophic bacteria in gramineous energy plants, Plant Soil

1997;194:45-55.

4. Chen, C., Bauske, E. M., Mussan, G., Rodriguez-Kabana, R. and Kloepper,

J. W.: Biological control of Fusarium wilt on cotton by use of endophytic

bacteria, Biol. Control, ;9955:83-91.

5. Liu, L., Kloepper, J. W. and Tuzun, S.: Induction of systemi resistance in

cucumber against Fusarium wilt by plant growth-promoting rhizobacteria,

PhytoPath, 1995;5:695-698.

6. Sturz, A. V.: The role of endophytic bacteria during seed piece decay and

potato tuberization, Plant Soil, 1995;175:257-263.

7. Frommel, M. I., Nowak, J. and Lazorovits, G.: Treatment of potato tubers

with a Pham Quang Hung et al. growth promoting Pseudomonas sp.: plant

growth responses and bacterium distribution in the rhizosphere, Plant Soil,

1993;150:51-60.

8. McInroy, J. A. and Kloepper, J. W.: Survey of indigenous bacterial

endophytes from cotton and sweet corn, Plant Soil, 1995;173:337- 342.

9. Carroll, G. C.: The Biology of Endophytism in Plants with Particular

Reference to Woody Perennials. In: Microbiology of Phyllosphere

(Fokkema, N., J. and van den Heuvel, J., eds.), Cambridge University

Press, London, 1986;205-222.

10. Petrini, O.: Fungal Endophyte of Tree Leaves. In: Microbial Ecology of

Leaves (Andrews J and Hirano SS, eds.), Spring-Verlag, New York,

1991;179-197.

11. Saikkonen, K., Faeth, S. H., Helander, M. and Sullivan T. J.: Fungal

endophytes: a continuum of interactions with host plants, Ann. Rev. Ecol.

Syst, 1998;29: 319-343.

12. Misaghi, I. J. and Donndelinger, C. R.: Endophytic bacteria in symptom-

free cotton plants, Phytopathology, 1900;80: 808-811.

13. Chanway, C. P.: Inoculation of tree roots with plant growth promoting soil

bacteria: an emerging technology for reforestation, Forest Sci, 1997;43: 99-

112.

14. Ting, A. S. Y., Meon, S., Kadir, J. and Radu, S.: Endophytic

microorganisms as potential growth promoters of banana, BioControl,

2008;53: 541-553.

15. Araújo, W. L., Marcon, J., Maccheroni, W. J. and Van Elsas, J. D.:

Diversity of endophytic bacterial populations and their interaction with

Xylella fastidiosa in citrus plants, Appl. Environ. Microbiol, 2002;68:

4906-4914.

16. Lacava, P. T., Araújo, W. L., Marcon, J. and Maccheroni, W. J.: Interaction

between endophytic bacteria from citrus plants and the phytopathogenic

bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis, Lett.

Appl. Microbiol, 2004;39: 55-59.

17. Yanni, Y. G., Rizk, R. Y., Corich, V., Squartini, A., Ninke, K., Philip-

Hollingsworth, S., Orgambide, G., Bruijn, F. D., Stoltzfus, R., Buckley, D.,

Schmidt, T., Mateos, P. F., Ladha, J. K. and Dazzo, F. B.: Natural

endophytic association between Rhizobium leguminosarum bv. trifolii and

rice roots and assessment of its potential to promote rice growth. Plant and

Soil, 1997;194: 99-114.

18. Mayak, S., Tirosh, T. and Glick, B. R.: Plant growth promoting bacteria

confer resistance in tomato plants to salt stress, Plant Physiology and

Biochemistry, 2004;42: 565-572.